sue in the embryo causes massive cell death in the neuronal populations that project to the target. These regressive events caused by the ablation experiments ...
Int. J. DH. Bioi. 39: 855-868 (1995)
855
Neurotrophins and their receptors in chicken neuronal development FINN HAllBOOK*, Department
ANDERS BACKSTROM, KlAS KUllANDER, REG WilLIAMS' and TED EBENDAl
of Developmental
Neuroscience,
Uppsala University,
Biomedical
ANNIKA KYlBERG, Center, Uppsafa,
Sweden
ABSTRACT A review on current studies of chicken neurotrophins and their receptors is given. Chicken NGF, BDNF and NT-3 have been cloned and sequences have been used to synthesize oligonucleotides for specific localization of expression during development. Also, chicken TrkA, TrkB and TrkC have been cloned, sequenced and studied by in situ hybridization. Recombinant NT3 was applied to chicken ganglia at different developmental stages to examine acquirement of responsiveness to NT-3 compared to NGF. Phylogenetic analyses of the chicken neurotrophins and Trk receptors were carried out based on parsimony. Finally, some data on apoptosis in chicken embryo sympathetic ganglia are presented.
KEY WORDS: avian, BDXF, evolution, NGF, NT-J, JetHor)', target-derived /aclm; Trk, tJ1vsine kinase reoptors
Introduction Neuronal death has been identified as a widely occurring phe-
nomenon in the development of the vertebrate nervous system. Hamburger and Levi.Montalcini (1949) demonstrated the extent of neuronal death during normal development in the nervous system. They found that marked degeneration of neurons occurred in chick dorsal root ganglia (DRG) around embryonic days 5 to 7. Subsequent work has shown that naturally occurring neuronal death is present in many neuronal cell populations both in the peripheral and central nervous systems. Examples include motoneurons (Oppenheim et al., 1982), ciliary neurons (Landmesser and Pilar, 1974) and sensory neurons of the chick (Hamburger et al., 1981). Naturally occurring neuronal death usually takes place during restricted time periods, as in the case of the chick ciliary ganglion where half of the neurons die between embryonic days E8 and E14 (Landmesser and Pilar, 1974)_The extent of the naturally occurring neuronal death can be up to 60% of the original number of neurons (Berg, 1982). Numerous studies have shown that removalof the target tissue in the embryo causes massive cell death in the neuronal populations that project to the target. These regressive events caused by the ablation experiments follow the same time course as the naturally occurring neuronal death (reviewed in Cowan ef al., 1984). The results indicated that the neurons acquire a dependence on the target tissue (Hamburger, 1977). Together, the data have led to a model with neurons that depend on target tissue or on factors secreted from the target. These factors mediating the survival effects were named neurotrophic factors. This
model was also supported by studies of the effect on neuronal survival after supplying additional target tissue. Transplantation of extra limbs to the embryonic tadpole before periods of naturally occurring neuronal death increased the number of surviving motoneurons in frog (Hollyday and Hamburger, 1976). The neuronal death has been shown to be an active process involving the mechanisms of apoptosis (Martin et al., 1988; Oppenheim et al., 1990; Garcia et al., 1992; Allsopp et a/., 1993). Nerve growth factor (NGF) has served, since it was discovered (Bueker, 1948; Levi-Montalcini and Hamburger, 1951; LeviMontalcini and Hamburger, 1953), as a prototype for the neurotrophic factors. NGF belongs to a family of four structurally related proteins known as neurotrophins, which support neuronal survival both in the developing and adult nervous system. In addition to NGF, the neurotrophin family includes brainderived neurotrophic factor (BDNF; Barde et a/., 1982; Leibrock et al., 1989), neurotrophin-3 (NT-3; Ernfors et al., 1990; Hohn et a/.,'1990; Jones and Reichardt, 1990; Kaisho et al., 1990; Maisonpierre et al_, 1990a; Rosenthal et al_, 1990) and neurotrophin-4 (NT-4; Hallbbbk et al., 1991; Ip et al., 1992) also known as neurotrophin-5 (Berkemeier et al., 1991). All four factors have similar binding characteristics to a low-affinity receptor (Ernfors et al., 1990; Rodriguez-Tebar et a/., 1990; Hallbbbk et al., 1991; Squinto et al., 1991), which is represented by a transmembrane glycoprotein of about 75 kDa (p75LNGFR) (Johnson et al., 1986; Radeke et al., 1987). The neurotrophins also bind
and activate a second class of receptors, which are tyrosine kinase receptors, known as the Trk receptors. NGF binds and activates the trk proto-oncogene product, which is a glycoprotein
-Address for reprints: Department of Developmental Neuroscience, Uppsala University, Box 587, Biomedical Center, 5-751 23 Uppsala. Sweden. FAX: 46.18.559017. 'Present address: Department of Molecular and Developmental Biology, Medical Nobel Institute, Karolinska Institute, S-171 77, Stockholm, Sweden. 0214-6282195/$03.00 CUBCPJn., PrintcdinSp;Un
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856
F. HallbtWk el al. Fig. 1. Schematic representation of a chicken E4.5 embryo. The shaded areas are parts of the avian developing nervous system. Indicated are cranial, sensory and future sympathetic ganglia. The plan of sectioning showed in Figs. 2 and 5 are indicated as XI and X2, respectively. ba: branchiat arches, Ib: leg bud ov: otic vesicle, spin: spinal sensory ganglia,
tributes to the high-affinity NGF binding including the TrkA receptor (Mahadeo et al., 1994; Verdi et al., 1994). In this report we review our current research and approaches to understand and illustrate the developmental mechanisms that include the actions of neurotrophins. We are using the chicken embryo as a model (Fig. 1), and molecular cloning of genes for the avian neurotrophins and their Trk receptors has allowed us to perform detailed comparative studies of the sites and patterns of synthesis of the corresponding mRNA.
symp: sympathetic
Results and Discussion
ganglia (future
position), trig: trigeminal ganglion, v-a: vestibula-acoustic ganglion,
Neurotrophins
are expressed
in the target fields of develop-
ing neurons of about 140 kDa. TrkA (Cordon et at., 1991; Hempstead et al., 1991; Kaplan et al., 1991a,b; Nebrada et al., 1991). There are two more TrkA-related tyrosine protein kinases, namely the 145 kDa glycoprotein gp145TrkB (Glass et al., 1991; Klein et al., 1991b; Soppet etal., 1991; Squintoetal" 1991)andthe 145 kDa glycoprotein gp145TrkC (Lamballe et al., 1991). These have been shown to constitute functional receptors for BDNF and NT3. respectively.The TrkB receptor has also been shown to be activated by NT-4 (Ip et al., 1992). The Trk tyrosine kinase receptors mediate biological responses of the neurotrophins by activating several signal transduction pathways which include regulators of the phosphatidyl inositol metabolism and the activity of ,., protein and Rat-1 proteins (Vetter et al., 1991; Loeb et the p21 al., 1992; Soltoff et al., 1992; Stephens et al., 1994). The function of the p75LNGFR is not fully clear for BDNF, NT-3 and NT4 but in the case of NGF, evidence is presented that it con-
NGF, BDNF and NT-3 have been cloned from the chicken (Ebendal et al., 1986; Hallb66k et al., 1991) and are likely to mediate a host of neurotrophic interactions in chicken development both in CNS and PNS. In particular we focus our attention here on peripheral ganglia in the deveioping chicken embryo. Neurons in the peripheral nervous system of the chicken embryo include sensory neurons located to cranial and spinal ganglia. The sensory innervation already starts soon after formation of the ganglia and neurites are extended towards their future terminal fields. Neurons in the lumbar sensory spinal ganglia innervate the developing limb buds. Both exteroceptive and proprioceptive sensory neurons in these ganglia have their future target areas in the developing buds, and in the E4 chicken embryo the ganglia are still close to the limb buds, and growth factors produced in the target can reach the neurites (Fig. 1). Using in situ hybridization analysis with probes for the neurotrophins, expres-
Fig. 2. In situ hybridization analysis of BDNF and trkB mRNA in the developing limb bud and spinal sensory ganglia. (AI Bright-field microphotograph showing in Situ analysis of transversal sections through the leg bud and lumbar region of an E4_5 chicken embryo. Boxed regions indicate the magnified areas in panel Band C. IB) Darkfield micrograph showing BDNF expression in the developing limb bud. IC} trkB mRNA expression in the spinal sensory ganglia. The trkB probe recognizes the mRNA encoding the catalytic trkB receptor. Ib: limb bud, n: notochord, sc: spinal cord, sp: spinal sensory ganglia. Scale bars: A, 300 tlm; B, 50 }1m; C, 50 j1m.
Neurotrophins
N
N N-linked glycosylation
.
siles 1_)
Cys 1
.
Leu
.
Cys II
Fig. 3. Schematic presentation of the chicken TrkA, TrkB and TrkC receptor isoforms. Overview of the chicken rrk isoforms sequenced and used for synthesis of ofigonucleatides applied to in situ hybridization analysis. Based on detected insertion sites in the TrkA extracellular region (Barker et al., 1993) and in kinase domain of TrkC (Lamballe et al.,
1993;
Valenzuela
Tsoulfas et al.,
et
al.,
N
seCluence
IgC2
,
IgC2
::DAII
Transmembrane~ domain
Tyrosine
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N
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1993) and in the
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frozen sections, BDNF mRNA is expressed in the E4.5 limb bud which corresponds to the target field for sensory neurons in the lumbar spinal ganglia. An adjacent section including the lumbar spinal ganglia hybridized with a probe for the mRNA encoding catalytic TrkB receptors shows that trkS mRNA expressing neurons are present in the ganglia. Neurons in the spinal ganglia have been shown to be supported by BDNF, and the trkB mRNA expressing neurons are most likely supported by BDNF during the period of naturally occurring neuronal death and wili respond with neurite outgrowth when BDNF protein is encountered. It has also been shown that NT-3 and NT-4 are expressed in the developing limb bud (Hallb66k et al., 1993; Henderson et al., 1993) and most probably NGF mRNA is expressed in the epithelium of the developing extremities (Wyatt et al., 1990). NT-4 still remains to be cloned and sequenced in the chick. In addition to the target field-derived mode of action of the neurotrophins, evidence suggesting a local, either autocrine or paracrine mode of action, is emerging. Thus, BDNF and NT-3 mRNA are expressed in embryonic sensory ganglia and that some of these ganglia contain cells that may express both the neurotrophin and its corresponding Ttk receptor (Ernfors and Persson, 1991; Schectetson and Bothwell, 1992). These results suggest a local action of the neurotrophins within both neural crest and placode-derived sensory neurons in the peripheral nsr-
-
.. .. ..
"""..""..
.,"",""""""",", " ,.,.",,,,..,..,,,,
gP145trkC
s10n of neurotrophin mRNA during the critical periods of innervation and cell death can be studied. Complex, but specific expression patterns for each of the neurotrophln mRNAs in the developing embryo are described and the spatia-temporal patterns are in many cases consistent with a target-derived mode of action where the neurotrophin mRNA is detected in the target fields of neuronal innervation, and mRNA for the corresponding Trk receptors are detected in the innervating neuron body. As shown in Figure 2, using in situ hybridization analysis on fresh
-
N
1993;
case of trkB in the extracellular portion (unpublished results) we were able to detect different isoforms of the receptors.
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857
TrkC
TrkB
Trk
Signal
i/1 chick development
-
-
-
t
Fig. 4.
Detection
of NT.3 mRNA
in developing
_
skin
of the
E4 chick-
en embryo head. (AI Section through rhe head of an E4 chicken embryo through the telencephalon, eyes and the nasal pits shown by bright-field microscopy afrer hybridization with the NT-3 probe. (81 Darkfield illumination and higher magnification of the boxed region in pane'
A showing
labeling for NT-3 mRNA in the developing skin. Epithelium (e), retina of the eye (r), telencephalon (r). Bars: A 250 pm; B. 100 J1m.
858
F. Hallbi;(jk et al.
I
Neurotrophills vous system. It remains to be determined how the local action of neurotrophins function together with a target-derived mode of action.
ill chick developmellt
859
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Neurotrophin receptors A strategy based on PCA and degenerate primers was used to isolate the chicken TrkA, TrkB and TrkC. An oligonucleotide probe for the trkB mANA (used in the analysis shown in Fig. 2C) is directed to the part of the trkB mANA encoding the intracellular tyrosine kinase domain of the receptor. This domain is essen. tial for signal transduction pathway activation. There are several isoforms of the Trk receptors with insertions and deletions in the extra- and intracellular domains (Fig. 3). Isoforms lacking the intracellular tyrosine.kinase domain have been characterized for TrkB and TrkC and these truncated receptors can bind neurotrophins but are believed to be unable to activate the signal transduction pathway (Klein et al., 1989; Middlemas et al., 1991). The system Is complex and TrkC isoforms have been found with amino-acid insertions in the tyrosine.kinase domain. These TrkC isoforms have different biological properties (Tsoulfas et al., 1993; Valenzuela et al., 1993) and substrate specificity (Lamballe et al., 1993). Using probes of 45 to 55 nucleotides, particular mRNA sequences for isoforms can be specifically detected. We have probes that are directed to the region of insertions in the tyrosine kinase domain spanning the insertion position and will therefore be specific for mANAs that encode catalytic TrkC receptors without insertions in the tyrosine kinase domain. It is not clear whether there are isoforms of the TrkA or TrkB receptors that have insertions in the tyrosine kinase domain but we have oligonucleotide probes for trkA and trkB mANA that are directed to the same region of the tyrosine kinase domain as the trkG probe. These probes will also be specific for catalytic TrkA and TrkB receptors (Fig. 3). Another example of a neuronal population that has been extensively examined is the sensory neurons in the trigeminal ganglion which innervate the skin and epithelium of the face and oral cavity (Fig. 1). Starting around E3, neurons in the newly formed trigeminal ganglion send out neurites into the upper branchial arches. In situ hybridization analysis with a probe for chicken NT-3 shows that NT-3 mANA is expressed at E4.5 in the epithelium which will be the future endothelium in the oral cavity and the facial skin (Fig. 4). As shown in several studies, the target fields of neurons in the trigeminal ganglion express NGF, BDNF and NT-4 mANA in addition to NT-3 mANA (Davies et al., 1986a, 1987; Ernfors etal., 1992; Buchmann and Davies, 1993; Hallbaak et al., 1993; Ibanez et al., 1993). In agreement with expression of all neurotrophins in the target fields for the neurons in the trigeminal ganglion, mANA encoding all three neurotrophin Trk receptors have been detected in the ganglion (Fig. 5; Ernfors etal., 1993; Ibanez etal., 1993; Williams etal., 1993, 1994). To analyze the distribution of neurons within the trigeminal sensory system, we have thus periormed in situ hybridization
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